L. Söderquist

Effect of cooling rates on post-thaw sperm motility, membrane integrity, capacitation status and fertility of dairy bull semen used for artificial insemination in sweden

We studied the effects of 2 different cooling rates during equilibration of semen from room temperature to 4 degrees C, at 4.2 degrees C/min (control split sample) or at 0.1 degree C/min (treatment split sample) on in vitro sperm viability post thawing and fertility after AI. Forty batches of split-frozen semen from 14 dairy bulls (Swedish Red and White breed) aged 14 to 16 m.o. or 66 to 79 m.o. were evaluated post-thawing for sperm motility (visual and computer-assisted sperm analysis [CASA], membrane integrity (fluorescent microscopy and flow cytometry post-loading with the combined fluorophores Calcein AM/EthD-1 and SYBR-14/PI); acrosomal status (with Pisum sativum agglutinin [PSA] staining); and capacitation status (CTC-assay). Fertility values (56-d nonreturn rate) of the slow cooling batches (treatment) were 0.4% units higher than for faster cooled (control) batches, but the difference was not statistically significant. Fertility values for the older bulls were 1.6% units higher than for the group of younger sires. No statistically significant correlations were found between semen viability parameters assessed in vitro and 56-d nonreturn rate. Visually assessed sperm motility, membrane integrity, capacitation and acrosomal status post-thawing did not differ significantly between cooling procedures, however the percentage of motile spermatozoa and the kinetic characteristics of spermatozoa--average path velocity (VAP), straight path velocity (VSL) and curvilinear velocity (VCL)--assessed by CASA differed significantly between cooling procedures. The results indicate that most of the in vitro sperm viability parameters post-thawing and the fertility results for bulls after AI did not differ significantly between the 2 semen cooling procedures tested.

Effect of different extenders and storage temperatures on sperm viability of liquid ram semen

Semen was collected with an artificial vagina from four adult rams. The ejaculates were pooled and diluted, using a split-sample technique, in four different extenders: one for milk (Mi), one for sodium citrate (Na), and two for Tris-based extenders (T1 and T2) including egg yolk. Thereafter, the diluted semen was stored at 5 and 20 degrees C, respectively. We evaluated sperm viability after 0, 6, 12, 24 and 30 h of storage. We assessed sperm motility subjectively, and we determined sperm membrane integrity using both the hypo-osmotic resistance test (ORT) and a fluorophore staining (SYBR-14 and propidium iodide) technique. We evaluated acrosomal status with Spermac and capacitation status with Chlortetracycline (CTC assay). All sperm viability parameters were influenced by storage time and extender, while sperm motility was the only evaluated parameter that was influenced by the interaction between extender and temperature. Semen that was diluted and stored in the commercially available Tris-based extender (T2) maintained sperm motility for a longer period of time, and acrosome and membrane integrity was higher during storage for up to 30 h as compared to the other extenders independent of storage temperature. In general, however, storage of ram semen at 5 degrees C seemed to influence sperm viability parameters less than storage at 20 degrees C. In conclusion, the results of the present study indicate that Tris-based extenders, especially T2, preserved sperm viability better than both the sodium citrate- and the milk-based extender did when liquid ram semen was stored up to 30 h at 5 and 20 degrees C. Whether the differences found between the extenders will be reflected in the fertility results after AI is yet unknown and needs to be further studied.

Assessment of sperm characteristics post-thaw and response to calcium ionophore in relation to fertility in Swedish dairy AI bulls

The present study examined the relationship between bull sperm characteristics post-thawing, after swim-up, and after challenge to calcium ionophore in relation to fertility (56-d nonreturn rates) after artificial insemination (AI). Spermatozoa from 25 semen batches derived from 15 Swedish Red and White AI bulls were evaluated with regard to post-thaw motility, membrane integrity, and migration through a swim-up procedure. The swim-up separated spermatozoa were assessed in terms of sperm concentration, viability and capacitation status as well as their response to exogenous calcium ionophore (A23187). Acrosome reactions were evaluated by fluorescence microscopy and flow cytometry. Sperm motility and viability post-thawing were significantly correlated with fertility. For the swim-up separated semen, significant correlations to nonreturn rates were found for concentration, viability, number of viable spermatozoa and sperm capacitation status (Pattern F and Pattern B). The only parameter significantly correlated to fertility after the ionophore challenge was the percentage of acrosome-reacted spermatozoa with remaining equatorial fluorescence, as assessed by fluorescence microscopy, but not by flow cytometry. The regression analysis showed that combining the results of sperm membrane integrity assessment post-thawing with those of capacitation status after swim-up provided the best prediction of fertility. The accuracy of prediction did not improve when these parameters were combined with the percentage of spermatozoa in which acrosome reaction was induced by ionophore challenge.

Breeding soundness evaluation of extensively managed bulls in Costa Rica

This paper describes the results of single breeding soundness evaluations (BSE) in 898 Bos indicus, Bos taurus and B. indicus x B. taurus bulls, 1 to 12 yr old, extensively reared in different climatic regions of Costa Rica and representing approximately 2% of the total breeding bull population. Thirty-three percent (n = 296) of the bulls were classified as unsound for breeding owing to clinical problems (9.1%, n = 82), low scrotal circumference (SC) being the most common finding, followed by unsatisfactory sperm morphology (23.9%, n = 214). The prevalence of bulls unsound for breeding was lowest in Bos indicus (29%, P < 0.01), intermediate in B. taurus (41%), and highest in B. indicus x B. taurus (48%). The percentages of abnormal sperm heads, acrosomes and midpieces tended to be higher in the ejaculates of bulls with softer testicular consistency (P < 0.001), a long scrotum (P < 0.01) or a low SC (P < 0.05), and such bulls were more often classified as being unsound for breeding (P < 0.05). Frequencies of sperm abnormalities were higher in bulls < 2 yr of age than in older males (P < 0.01) and were highest in B. indicus x B. taurus bulls (P < 0.001). The results confirm differences between species in their adaptability to a tropical environment and support earlier evidence of an association between SC, testicular consistency and scrotal length clinical parameters, and testicular function in bulls.

Influence of season, age, breed and some other factors on the variation in sperm morphological abnormalities in Swedish dairy A.I. bulls

Abstract Fifty-two dairy A.I. bulls aged from 14 months to 6 and a 1 2 years old were used to study the effects of age, breed, season and, dehorning and dihydrostreptomycin treatments on the incidence of sperm morphological abnormalities in fresh semen. The highest mean value of sperm abnormalities was recorded for abnormal heads (6.1%), but most of the recorded abnormalities only occurred in approximately 1–2% of the studied spermatozoa. Significant seasonal variation occurred in the incidences of sperm head abnormalities and total sperm abnormalities. Least-squares means for sperm abnormalities were significantly higher during the warmer seasons (spring, summer) compared with the colder ones (autumn, winter). The incidence of sperm abnormalities (abnormal heads, proximal droplets, total abnormalities) was relatively constant over time within bull (repeatability estimates varied between 0.36 and 0.54), but was significantly affected by age (abnormal heads and acrosomes, total abnormalities) and breed (abnormal heads, detached heads). The incidence of total sperm abnormalities was also found to be significantly increased in semen from a group of bulls submitted to both dehorning and double injections of dihydrostreptomycin (DHS) when comparing pre- and posttreatment periods. The results indicate that age, breed and seasonal effects as well as repeated stressful treatments influence the sperm characteristics and must be considered when evaluating bulls for sperm morphological traits.

Assessment of ram sperm membrane integrity following different thawing procedures

Semen from five 2.5-yr-old rams selected for use in an AI program was collected over 3 consecutive days using an artificial vagina. The semen was diluted with a skim milk extender containing 7% glycerol (v/v), packed in French mini-straws (approx. 100 mill/straw), and frozen in a programmable freezer. Three freezing operations were carried out per ram. Three straws per freezing operation were subjected to the following thawing procedures: 1) 70 degrees C, 5 sec; 2) 50 degrees C, 9 sec and 3) 35 degrees C, 12 sec. Post-thaw sperm motility was subjectively assessed using a phase contrast microscope; while the combined fluorochromes carboxyfluorescein diacetate and propidium iodide (CFDA/PI), the hypo-osmotic swelling test (HOS) and the presence of normal apical ridges (NARs) were used to determine the degree of sperm membrane integrity. Significant differences between thawing treatments were found for post-thaw motility (P < .05) and membrane integrity (P < 0.01), and variation among rams was statistically significant. Post-thaw sperm motility as well as the percentage of spermatozoa showing intact membranes were significantly higher (P < 0.01) for straws thawed at 70 degrees C than for those thawed at 35 degrees C (67.0 +/- 1.1 and 63.0 +/- 1.1%, and 50.5 +/- 1.5 and 41.7 +/- 1.5%, respectively). However, no corresponding statistically significant difference could be found for these parameters when 70 degrees C and 50 degrees C thawing were compared. It was concluded that sperm can be thawed at 50 degrees C for 9 sec instead of 70 degrees C for 5 sec without further reducing sperm motility or membrane integrity. This lower thawing temperature would facilitate the widespread use of frozen/thawed ram semen under farm conditions in Sweden.

Effect of milk- and TRIS-based extenders on the fertility of sheep inseminated vaginally once or twice with liquid semen

We studied the influence of two different extenders, a milk-based versus a TRIS-based extender, using a split-sample technique, on fertility after single and double vaginal inseminations in natural estrous in Norwegian Crossbred ewes. Semen from 21 Norwegian Crossbred rams, all aged approximately 0.5 years, was used for AI of totally 561 Norwegian Crossbred ewes housed at 37 different farms. The farmers performed the inseminations themselves. The ewes were allocated to four parallel groups based on the two extenders and single or double inseminations (2 x 2). The farmers were recommended to inseminate the ewes between 12 and 24 h after detection of natural standing estrous. Vaginal insemination with cooled liquid semen diluted in the milk-based extender resulted in a statistically significant (P<0.01) better fertility of about 10% units both as 25-day NR (non return rate)-and lambing rates, compared with semen diluted in the TRIS-based extender. Double inseminations gave significantly higher (P=0.03) fertility results for both extenders expressed as 25-day NR results, but was not quite statistically significant when expressed as lambing rates (P=0.06) compared with single insemination. The overall 25-day NR results for the milk-based extender (66.4%) after single inseminations is in accordance with both the national results (67.1%) based on vaginal inseminations of 11,377 ewes, as well as with the results from a previous study in the same region achieving a 25-day NR results of 63.3%. In conclusion, liquid ram semen diluted in a milk-based extender and vaginally inseminated once in natural heat, with a semen dose of 150 x 10(6) spermatozoa, gave acceptable fertility results and is to be recommended as the method of choice in Norway.

Effect of vaginal and cervical deposition of semen on the fertility of sheep inseminated with frozen-thawed semen

The effect of vaginal and cervical deposition of frozen-thawed semen on the fertility of sheep was tested in a field trial in which 543 Norwegian crossbred ewes aged between six months and five-and-a-half years from 10 farms were inseminated after natural oestrus. Cervical insemination with 200 x 106 spermatozoa resulted in 25-day non-return and lambing rates of 75·4 and 72·7 per cent, respectively, and vaginal insemination gave rates of 71·3 and 67·4 per cent; the cervical inseminations produced significantly higher lambing rates (P=0·04). There were significant differences between the lambing rates for different rams (P=0·006) and different farmers (P=0·003), and there was a significant interaction between farmer and deposition site (P=0·03). After vaginal insemination fertility was encouragingly high, but the results varied with the farmer, and different flock and management conditions.

Effect of deposition site and sperm number on the fertility of sheep inseminated with liquid semen.

The effect of the deposition site and the numbers of sperm on the fertility of sheep was tested in a field trial in which 1292 Norwegian crossbred ewes aged between six months and five-and-a-half years from 52 farms were inseminated with liquid semen after natural oestrus. Cervical insemination with 150 x 106 and 75 x 106 spermatozoa resulted in 25-day non-return rates of 63.7 and 56.1 per cent, and vaginal insemination gave non-return rates of 63.3 and 56.6 per cent, respectively. There was no significant difference between the cervical and vaginal inseminations, but the inseminations with 150 x 106 spermatozoa gave significantly higher non-return rates (P=0.004). There were significant differences between the non-return rates for different rams (P<0.0001) and farmers (P=0.0002) but the age of the ewe had no significant effect.

Joint disorder; a contributory cause to reproductive failure in beef bulls?

The lame sire, unsound for breeding, can cause substantial economic loss due to reduced pregnancies in the beef-producing herd.To test the hypothesis that joint disorder is a possible cause of infertility in beef sires, right and left hind limb bones from 34 beef sires were examined postmortem to identify lesions in the femorotibial, femoropatellar (stifle), tarsocrural, talocalcaneus, and proximal intertarsal (tarsal) joints. The bulls were slaughtered during or after the breeding season due to poor fertility results. Aliquots of the cauda epididymal contents taken postmortem from 26 bulls were used for sperm morphology evaluation. As a control, hind limbs (but no semen samples) from 11 beef bulls with good fertility results were included.Almost all infertile bulls (30/34) had lesions in at least one joint. Twenty-eight bulls (28/30, 93%) had lesions in the stifle joint, and 24 (24/28, 86%) of these were bilateral. Fourteen bulls (14/30, 47%) had lesions in the tarsal joint, and 10 (10/14, 71%) of these were bilateral. Four bulls (4/34, 12%) had no lesions, three bulls (3/34, 9%) had mild osteoarthritis (OA), 5 (5/34, 15%) moderate OA, 17 (17/34, 50%) severe OA and 5 (5/34, 15%) deformed OA. Almost all OA lesions (97%) were characterized as lesions secondary to osteochondrosis dissecans. All the bulls with satisfactory sperm morphology (n = 12/34) had joint lesions, with mostly severe or deformed bilateral lesions (83%). Consequently, the most likely cause of infertility in these 12 bulls was joint disease. Almost all control bulls (10/11) had OA lesions, but most of them were graded as mild (55%) or moderate (36%). None of the control bulls had severe lesions or deformed OA.We suggest that joint lesions should be taken into consideration as a contributory cause of reproductive failure in beef sires without symptoms of lameness.